Lettuce named spritzer

ABSTRACT

Novel lettuce cultivar, designated SPRITZER is disclosed. The invention relates to the seeds of lettuce cultivar SPRITZER, to the plants of lettuce line SPRITZER and to methods for producing a lettuce plant by crossing the cultivar SPRITZER with itself or another lettuce line, respectively. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce lines derived from the cultivar SPRITZER.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 14/577,243, filed Dec. 19, 2014, which claims priority to, andthe benefit of U.S. Provisional Patent Application No. 61/929,606, filedJan. 21, 2014, each of which is herein incorporated by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to new and distinctive lettuce (Lactucasativa) cultivars, designated SCARAMANGA, GIMLET, SPRITZER, and/orMOJITO.

BACKGROUND OF THE INVENTION

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.

In lettuce, these important traits may include increased head size andweight, higher seed yield, improved color, resistance to diseases andinsects, tolerance to drought and heat, better post-harvest shelf-lifeof the leaves, better standing ability in the field, better uniformity,and better agronomic quality.

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa which is grown for its edible head and leaves. Asa crop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield. Lettuce is theworld's most popular salad. In the United States, the principal growingregions are California and Arizona which produce approximately 329,000acres out of a total annual acreage of more than 333,000 acres (USDA,2005). Fresh lettuce is available in the United States year-roundalthough the greatest supply is from May through October. For plantingpurposes, the lettuce season is typically divided into three categories,early, mid and late, with the coastal areas planting from January toAugust, and the desert regions from August to December. Lettuce isconsumed nearly exclusively as fresh, raw product, and occasionally as acooked vegetable. Baby leaf or spring mix lettuce is an increasinglypopular crop as worldwide baby leaf lettuce consumption continues toincrease. Spring mix lettuce refers to lettuce that is grown in highconcentrations and harvested at a very young or ‘baby leaf’ stage,typically 30 to 45 days after planting. The plantings are often done onwider 80 to 84 inch beds and often contain up to one million plants peracre. Compared to iceberg or romaine plantings, where they are typicallyharvested 60 to 100 days after planting, with a population of roughly25,000 to 30,000 plants per acre. Spring mix plantings often includemultiple types of lettuces, all harvested when the leaves are young andtender. These plantings can include green romaine, red romaine, darklolla rossa, tango, green leaf, and red leaf types. Spring mix fieldsare most often harvested mechanically and the harvested leaves arepacked in plastic totes, where they are transported to a processingfacility where they are washed, processed and mixed according to thesalad recipe.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositaefamily). Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are several morphological types of lettuce. TheCrisphead group includes the Iceberg and Batavian types. Iceberg lettucehas a large, firm head with a crisp texture and a white or creamy yellowinterior. Batavian lettuce predates Iceberg lettuce and has a smallerand less firm head. The Butterhead group has a small, soft head with analmost oily texture. Romaine lettuce, also known as Cos lettuce, haselongated upright leaves forming a loose, loaf-shaped head and the outerleaves are usually dark green. Leaf lettuce comes in many varieties,none of which form a head. There are three types of lettuce which areseldom seen in the United States: Latin lettuce, which looks like across between Romaine and Butterhead; Stem lettuce, which has long,narrow leaves and thick, edible stems; and Oilseed lettuce, which is aprimitive type of lettuce grown for its large seeds that are pressed toobtain oil.

Lactuca sativa is a simple diploid species with nine pairs ofchromosomes (2N=18). Lettuce is an obligate self-pollinating specieswhich means that pollen is shed before stigma emergence, assuring 100%self-fertilization. Since each lettuce flower is an aggregate of about10-20 individual florets (typical of the Compositae family), manualremoval of the anther tubes containing the pollen is tedious. As aresult, a modified method of misting to wash off the pollen prior tofertilization is needed to assure crossing or hybridization. Flowers tobe used for crossings are selected about 60-90 minutes after sunrise.Selection criteria include plants with open flowers, where the stigmahas emerged and pollen is visibly attached to a single stigma (there areabout 10-20 stigma). Pollen grains are washed off using 3-4 pumps ofwater from a spray bottle and with enough pressure to dislodge thepollen grains without damaging the style. Excess water is then dried offusing clean paper towels and about 30 minutes later, the styles springback up and the two lobes of the stigma are visibly open in a “V” shape.Pollen from another variety or donor parent is then introduced by gentlyrubbing the stigma and style of the donor parent to the maternal parent.Most pertinent information including dates and pedigree are then securedto the flowers using tags.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, pure linecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location may be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcross breeding.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars.Nevertheless, it is also suitable for the adjustment and selection ofmorphological character, color characteristics and simply inheritedquantitative characters. Various recurrent selection techniques are usedto improve quantitatively inherited traits controlled by numerous genes.The use of recurrent selection in self-pollinating crops depends on theease of pollination, the frequency of successful hybrids from eachpollination and the number of hybrid offspring from each successfulcross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars. Those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and/or to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique andsuperior lettuce cultivars. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Another method used to developnew and unique lettuce cultivar occurs when the lettuce breeder selectsand crosses parental varieties followed by haploid induction andchromosome doubling that result in the development of dihaploidcultivars. The breeder can theoretically generate billions of differentgenetic combinations via crossing, selfing and mutations and the same istrue for the utilization of the dihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions and further selections arethen made during and at the end of the growing season. The cultivarsthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. This unpredictability results in theexpenditure of large amounts of research monies to develop superior newlettuce cultivars.

The development of new lettuce cultivars requires the development andselection of lettuce varieties, the crossing of these varieties and theevaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars of desired phenotypesare developed by selfing and selection or through the dihaploid breedingmethod.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents that possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals may beginin the F₂ population; then, beginning in the F₃, the best individuals inthe best families are selected. Replicated testing of families, orhybrid combinations involving individuals of these families, oftenfollows in the F₄ generation to improve the effectiveness of selectionfor traits with low heritability. At an advanced stage of inbreeding(i.e., F₆ and F₇), the best lines or mixtures of phenotypically similarlines are tested for potential release as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, lettuce breeders commonly harvest one ormore flower containing seed from each plant in a population and threshthem together to form a bulk. Part of the bulk is used to plant the nextgeneration and part is put in reserve. The procedure has been referredto as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than to remove one seed from each by hand for thesingle-seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPD5),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs-which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen (Molecular Linkage Map ofSoybean (Glycine max), pp. 6.131-6.138 in S. J. O'Brien (ed.) GeneticMaps: Locus Maps of Complex Genomes, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1993)) developed a molecular geneticlinkage map that consisted of 25 linkage groups with about 365 RFLP, 11RAPD, three classical markers, and four isozyme loci. See also,Shoemaker, R. C., RFLP Map of Soybean, pp. 299-309, in Phillips, R. L.and Vasil, I. K. (eds.), DNA-Based Markers in Plants, Kluwer AcademicPress, Dordrecht, the Netherlands (1994).

SSR technology is an efficient and practical marker technology; moremarker loci can be routinely used and more alleles per marker locus canbe found using SSRs in comparison to RFLPs. For example, Diwan andCregan described a highly polymorphic microsatellite locus in soybeanwith as many as 26 alleles. Diwan, N. and Cregan, P. B., Theor. Appl.Genet., 95:22-225 (1997). SNPs may also be used to identify the uniquegenetic composition of the invention and progeny varieties retainingthat unique genetic composition. Various molecular marker techniques maybe used in combination to enhance overall resolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid, or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company (1993). The production of doublehaploids can also be used for the development of homozygous varieties ina breeding program. Double haploids are produced by the doubling of aset of chromosomes from a heterozygous plant to produce a completelyhomozygous individual. For example, see Wan, et al., Theor. Appl.Genet., 77:889-892 (1989).

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Lettuce is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding lettucecultivars that are agronomically sound. The reasons for this goal areobviously to maximize the amount of yield produced on the land. Toaccomplish this goal, the lettuce breeder must select and developlettuce plants that have the traits that result in superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

According to the invention, there is provided four novel lettucecultivars designated SCARAMANGA, GIMLET, SPRITZER and/or MOJITO. Thisinvention thus relates to the seeds of lettuce cultivars SCARAMANGA,GIMLET, SPRITZER and/or MOJITO, to the plants, or part(s) thereof oflettuce cultivars SCARAMANGA, GIMLET, SPRITZER and/or MOJITO, to plantsor part(s) thereof consisting essentially of the phenotypic andmorphological characteristics of lettuce cultivars SCARAMANGA, GIMLET,SPRITZER and/or MOJITO, and/or having all the phenotypic andmorphological characteristics of lettuce cultivars SCARAMANGA, GIMLET,SPRITZER and/or MOJITO, and/or having the phenotypic and morphologicalcharacteristics of lettuce cultivars SCARAMANGA, GIMLET, SPRITZER and/orMOJITO listed in Tables 1 to 4, including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions. The invention also relates to variants,mutants and trivial modifications of the seed or plant of lettucecultivars SCARAMANGA, GIMLET, SPRITZER and/or MOJITO. Plant parts of thelettuce cultivars of the present invention are also provided such as,i.e., pollen obtained from the plant cultivars and an ovule obtainedfrom the plant cultivars.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act, i.e., a variety that: (i) is predominantlyderived from lettuce cultivars SCARAMANGA, GIMLET, SPRITZER and/orMOJITO or from a variety that is predominantly derived from lettucecultivars SCARAMANGA, GIMLET, SPRITZER and/or MOJITO, while retainingthe expression of the essential characteristics that result from thegenotype or combination of genotypes of lettuce cultivars SCARAMANGA,GIMLET, SPRITZER and/or MOJITO;

(ii) is clearly distinguishable from lettuce cultivars SCARAMANGA,GIMLET, SPRITZER and/or MOJITO; and

-   -   (iii) except for differences that result from the act of        derivation, conforms to the initial variety in the expression of        the essential characteristics that result from the genotype or        combination of genotypes of the initial variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivars SCARAMANGA, GIMLET, SPRITZERand/or MOJITO. The tissue culture will preferably be capable ofregenerating plants consisting essentially of the phenotypic andmorphological characteristics of lettuce cultivars SCARAMANGA, GIMLET,SPRITZER and/or MOJITO, and/or having all the phenotypic andmorphological characteristics of lettuce cultivars SCARAMANGA, GIMLET,SPRITZER and/or MOJITO, and/or having the physiological andmorphological characteristics of lettuce cultivars SCARAMANGA, GIMLET,SPRITZER and/or MOJITO listed in Tables 1 to 4, including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions. Preferably, the cells of such tissueculture will be embryos, meristematic cells, seeds, callus, pollen,leaves, anthers, pistils, roots, root tips, flowers, stems and axillarybuds. Protoplasts produced from such tissue culture are also included inthe present invention. The lettuce shoots, roots and whole plantsregenerated from the tissue culture are also part of the invention.

Also included in the invention are methods for producing a lettuce plantproduced by crossing lettuce cultivars SCARAMANGA, GIMLET, SPRITZERand/or MOJITO, respectively with itself or another lettuce cultivar.When crossed with itself, i.e., when SCARAMANGA is crossed with anotherlettuce cultivar SCARAMANGA plant or self-pollinated, when GIMLET, iscrossed with another lettuce cultivar GIMLET plant or self-pollinated,when SPRITZER is crossed with another lettuce cultivar SPRITZER plant orself-pollinated, when MOJITO is crossed with another lettuce cultivarMOJITO plant or self-pollinated, lettuce cultivar SCARAMANGA, GIMLET,SPRITZER and/or MOJITO, respectively will be conserved (e.g., as aninbred). When crossed with another, different lettuce plant, an F₁hybrid seed is produced. F₁ hybrid seeds and plants produced by growingsaid hybrid seeds are included in the present invention. A method forproducing an F₁ hybrid lettuce seed comprising crossing a lettucecultivar SCARAMANGA, GIMLET, SPRITZER and/or MOJITO plant with adifferent lettuce plant and harvesting the resultant hybrid lettuce seedare also part of the invention. The hybrid lettuce seed produced by themethod comprising crossing a lettuce cultivar SCARAMANGA, GIMLET,SPRITZER and/or MOJITO plant with a different lettuce plant andharvesting the resultant hybrid lettuce seed, are included in theinvention, as are the hybrid lettuce plant, or part(s) thereof, andseeds produced by growing said hybrid lettuce seed.

In another aspect, the present invention provides transformedSCARAMANGA, GIMLET, SPRITZER and/or MOJITO lettuce cultivar plants orpart(s) thereof that have been transformed so that its genetic materialcontains one or more transgenes, preferably operably linked to one ormore regulatory elements. Also, the invention provides methods forproducing a lettuce plant containing in its genetic material one or moretransgenes, preferably operably linked to one or more regulatoryelements, by crossing transformed SCARAMANGA, GIMLET, SPRITZER and/orMOJITO lettuce cultivar plants with either a second plant of anotherlettuce cultivar, or a non-transformed SCARAMANGA, GIMLET, SPRITZERand/or MOJITO lettuce cultivar, so that the genetic material of theprogeny that results from the cross contains the transgene(s),preferably operably linked to one or more regulatory elements. Theinvention also provides methods for producing a lettuce plant thatcontains in its genetic material one or more transgene(s), wherein themethod comprises crossing the cultivars SCARAMANGA, GIMLET, SPRITZERand/or MOJITO with a second lettuce cultivar of another lettuce cultivarwhich contains one or more transgene(s) operably linked to one or moreregulatory element(s) so that the genetic material of the progeny thatresults from the cross contains the transgene(s) operably linked to oneor more regulatory element(s). Transgenic lettuce cultivars, or part(s)thereof produced by the methods are in the scope of the presentinvention.

More specifically, the invention comprises methods for producing a malesterile lettuce plant, an herbicide resistant lettuce plant, an insectresistant lettuce plant, a disease resistant lettuce plant, a waterstress tolerant lettuce plant, a heat stress tolerant lettuce plant, anda lettuce plant with improved shelf-life and a lettuce plant withdelayed senescence. Said methods comprise transforming lettuce cultivarsSCARAMANGA, GIMLET, SPRITZER and/or MOJITO plant with a nucleic acidmolecule that confers, for example, male sterility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, heat stress tolerance, improved shelf life or delayedsenescence respectively. The transformed lettuce plants, or part(s)thereof, obtained from the provided methods, including, for example, amale sterile lettuce plant, an herbicide resistant lettuce plant, aninsect resistant lettuce plant, a disease resistant lettuce plant, alettuce plant tolerant to water stress, a lettuce plant tolerant to heatstress, a lettuce plant with improved shelf-life, a lettuce plant withimproved shelf-life and delayed senescence are included in the presentinvention. For the present invention and the skilled artisan, disease isunderstood to be fungal diseases, viral diseases, bacterial diseases orother plant pathogenic diseases and a disease resistant plant willencompass a plant resistant to fungal, viral, bacterial and other plantpathogens.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into lettuce cultivarsSCARAMANGA, GIMLET, SPRITZER and/or MOJITO and plants obtained from suchmethods. The desired trait(s) may be, but not exclusively, a singlegene, preferably a dominant but also a recessive allele. Preferably, thetransferred gene or genes will confer such traits as male sterility,herbicide resistance, insect resistance, resistance to bacterial,fungal, or viral disease, increased leaf number, improved shelf-life,delayed senescence and tolerance to water stress or heat stress. Thegene or genes may be naturally occurring gene(s) or transgene(s)introduced through genetic engineering techniques. The method forintroducing the desired trait(s) is preferably a backcrossing processmaking use of a series of backcrosses to lettuce cultivars SCARAMANGA,GIMLET, SPRITZER and/or MOJITO during which the desired trait(s) ismaintained by selection.

When using a transgene, the trait is generally not incorporated intoeach newly developed line/cultivar such as lettuce cultivars SCARAMANGA,GIMLET, SPRITZER and/or MOJITO by direct transformation. Rather, themore typical method used by breeders of ordinary skill in the art toincorporate the transgene is to take a line already carrying thetransgene and to use such line as a donor line to transfer the transgeneinto the newly developed line. The same would apply for a naturallyoccurring trait or one arising from spontaneous or induced mutations.The backcross breeding process of SCARAMANGA comprises the followingsteps: (a) crossing lettuce cultivar SCARAMANGA plants with plants ofanother cultivar that comprise the desired trait(s); (b) selecting theF₁ progeny plants that have the desired trait(s); (c) crossing theselected F₁ progeny plants with lettuce cultivar SCARAMANGA plants toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait(s) and physiological andmorphological characteristics of lettuce cultivar SCARAMANGA to produceselected backcross progeny plants; and (e) repeating steps (c) and (d)one, two, three, four, five six, seven, eight, nine, or more times insuccession to produce selected, second, third, fourth, fifth, sixth,seventh, eighth, ninth, or higher backcross progeny plants that consistessentially of the phenotypic and morphological characteristics oflettuce cultivar SCARAMANGA, and/or have all the phenotypic andmorphological characteristics of lettuce cultivar SCARAMANGA, and/orhave the desired trait(s) and the physiological and morphologicalcharacteristics of lettuce cultivar SCARAMANGA as determined in Table 1,including but not limited to at a 5% significance level when grown inthe same environmental conditions. The backcross breeding process ofGIMLET comprises the following steps: (a) crossing lettuce cultivarGIMLET plants with plants of another cultivar that comprise the desiredtrait(s); (b) selecting the F₁ progeny plants that have the desiredtrait(s); (c) crossing the selected F₁ progeny plants with lettucecultivar GIMLET plants to produce backcross progeny plants; (d)selecting for backcross progeny plants that have the desired trait(s)and physiological and morphological characteristics of lettuce cultivarGIMLET to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) one, two, three, four, five six, seven, eight, nine,or more times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth, or higher backcross progeny plantsthat consist essentially of the phenotypic and morphologicalcharacteristics of lettuce cultivar GIMLET, and/or have all thephenotypic and morphological characteristics of lettuce cultivar GIMLET,and/or have the desired trait(s) and the physiological and morphologicalcharacteristics of lettuce cultivar GIMLET as determined in Table 2,including but not limited to at a 5% significance level when grown inthe same environmental conditions. The backcross breeding process ofSPRITZER comprises the following steps: (a) crossing lettuce cultivarSPRITZER plants with plants of another cultivar that comprise thedesired trait(s); (b) selecting the F₁ progeny plants that have thedesired trait(s); (c) crossing the selected F₁ progeny plants withlettuce cultivar SPRITZER plants to produce backcross progeny plants;(d) selecting for backcross progeny plants that have the desiredtrait(s) and physiological and morphological characteristics of lettucecultivar SPRITZER to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) one, two, three, four, five six, seven,eight, nine, or more times in succession to produce selected, second,third, fourth, fifth, sixth, seventh, eighth, ninth, or higher backcrossprogeny plants that consist essentially of the phenotypic andmorphological characteristics of lettuce cultivar SPRITZER, and/or haveall the phenotypic and morphological characteristics of lettuce cultivarSPRITZER, and/or have the desired trait(s) and the physiological andmorphological characteristics of lettuce cultivar SPRITZER as determinedin Table 3, including but not limited to at a 5% significance level whengrown in the same environmental conditions. The backcross breedingprocess of MOJITO comprises the following steps: (a) crossing lettucecultivar MOJITO plants with plants of another cultivar that comprise thedesired trait(s); (b) selecting the F₁ progeny plants that have thedesired trait(s); (c) crossing the selected F₁ progeny plants withlettuce cultivar MOJITO plants to produce backcross progeny plants; (d)selecting for backcross progeny plants that have the desired trait(s)and physiological and morphological characteristics of lettuce cultivarMOJITO to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) one, two, three, four, five six, seven, eight, nine,or more times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth, or higher backcross progeny plantsthat consist essentially of the phenotypic and morphologicalcharacteristics of lettuce cultivar MOJITO, and/or have all thephenotypic and morphological characteristics of lettuce cultivar MOJITO,and/or have the desired trait(s) and the physiological and morphologicalcharacteristics of lettuce cultivar MOJITO as determined in Table 4,including but not limited to at a 5% significance level when grown inthe same environmental conditions. The lettuce plants produced by themethods are also part of the invention. Backcrossing breeding methods,well-known for one skilled in the art of plant breeding, will be furtherdeveloped in subsequent parts of the specification.

In a preferred embodiment, the present invention provides methods forincreasing and producing lettuce cultivar SCARAMANGA, GIMLET, SPRITZERand/or MOJITO seed, whether by crossing a first parent lettuce cultivarplant with a second parent lettuce cultivar plant and harvesting theresultant lettuce seed, wherein both said first and second parentlettuce cultivar plant are the lettuce cultivars SCARAMANGA, GIMLET,SPRITZER and/or MOJITO, respectively or by planting a lettuce seed ofthe lettuce cultivars SCARAMANGA, GIMLET, SPRITZER and/or MOJITO,growing a lettuce cultivar SCARAMANGA, GIMLET, SPRITZER and/or MOJITOplant from said seed, respectively, controlling a self pollination ofthe plant where the pollen produced by a grown lettuce cultivarSCARAMANGA plant pollinates the ovules produced by the very same lettucecultivar SCARAMANGA grown plant, where the pollen produced by a grownlettuce cultivar GIMLET plant pollinates the ovules produced by the verysame lettuce cultivar GIMLET grown plant, where the pollen produced by agrown lettuce cultivar SPRITZER plant pollinates the ovules produced bythe very same lettuce cultivar SPRITZER grown plant, where the pollenproduced by a grown lettuce cultivar MOJITO plant pollinates the ovulesproduced by the very same lettuce cultivar MOJITO grown plant, andharvesting the resultant seed.

The invention further provides methods for developing lettuce cultivarsin a lettuce breeding program using plant breeding techniques includingrecurrent selection, backcrossing, pedigree breeding, molecular markers(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs). AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, etc.) enhancedselection, genetic marker enhanced selection, and transformation. Seeds,lettuce plants, and part(s) thereof produced by such breeding methodsare also part of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Big Vein virus. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. An Oomycete that causes downy mildew in lettuce incooler growing regions.

Core length. Length of the internal lettuce stem measured from the baseof the cut and trimmed head to the tip of the stem.

Corky root. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Immunity to disease(s) and or insect(s). A lettuce plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Intermediate/Moderate resistance to disease(s) and or insect(s). Alettuce plant that restricts the growth and development of specificdisease(s) and or insect(s), but may exhibit a greater range of symptomsor damage compared to high/standard resistant plants. Intermediateresistant plants will usually show less severe symptoms or damage thansusceptible plant varieties when grown under similar environmentalconditions and/or specific disease(s) and or insect(s) pressure, but mayhave heavy damage under heavy pressure. Intermediate resistant lettuceplants are not immune to the disease(s) and or insect(s).

Maturity Date. Maturity refers to the stage when plants are of full sizeor optimum weight, and in marketable form or shape to be of commercialor economic value. In leaf types they range from 50-75 days from time ofseeding, depending upon the season of the year. In leaf types they rangefrom 65-105 days from time of seeding, depending upon the season of theyear

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Lettuce Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe lettuce at harvest.

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture, or incorporated in a plant or plantpart.

Plant part. As used herein, the term “plant part” includes any part ofthe plant including but not limited to leaves, heads, stems, roots,seed, embryos, pollen, ovules, flowers, root tips, anthers, tissue,cells, axillary buds, and the like.

Ratio of head height/diameter. Head height divided by the head diameteris an indication of the head shape; <1 is flattened, 1=round, and >1 ispointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

High/standard resistance to disease(s) and or insect(s). A lettuce plantthat restricts highly the growth and development of specific disease(s)and or insect(s) under normal disease(s) and or insect(s) attackpressure when compared to susceptible plants. These lettuce plants canexhibit some symptoms or damage under heavy disease(s) and or insect(s)pressure. Resistant lettuce plants are not immune to the disease(s) andor insect(s).

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

Susceptible to disease(s) and or insect(s). A lettuce plant that issusceptible to disease(s) and or insect(s) is defined as a lettuce plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses. A lettuce plant that is tolerant toabiotic stresses such as, for example bolting or tipburn has the abilityto endure abiotic stress without serious consequences for growth,appearance and yield.

Tip burn. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

Tomato Bushy Stunt. A disease which causes stunting of growth, leafmottling, and deformed or absent fruit.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar SCARAMANGA has superior characteristics and wasdeveloped from an initial cross that was made in a greenhouse in Gilroy,Calif. during the first year of development under number 71030040,between red romaine and red little gem plants. The F₁ plants were grownin Gilroy, Calif. F2 and F3 were selected for color improvement and anF3 plant showing slower bolting was taken from the F3 population toproduce an F4 seed. This plant also had a darker red leaf color than allother plants in the population. Selection for a slower bolting red leaftype with a heavy plant type continued through the F8 generation whichwas trialed as a baby leaf product.

Cultivar SCARAMANGA is a red leaf lettuce similar to lettuce cultivarsBlackhawk and Showdown. While somewhat similar to Blackhawk andShowdown, Cultivar SCARAMANGA produces a leaf with less undulation and amore entire leaf margin than the varieties Blackhawk and Showdown. Itsred color is similar to Blackhawk, but it has improved Bremia lactucaeresistance. Its mildew resistance is similar to Showdown, but it islighter in color and faster growing than Showdown, which is a benefitfor winter production.

Cultivar SCARAMANGA is a year-round, mildew resistant, red-leaf lettucevariety suitable for baby-leaf salad production in coastal California,the U.S. desert southwest, northern Europe, and southern Europe. It hasdark red, glossy leaf, and resistance to Bremia lactucae(CAI-VI/BL1-30).

Some of the criteria used to select in various generations include:Bremia resistance, slow bolting and red leaf color.

Lettuce cultivar SCARAMANGA shown uniformity and stability for thetraits, as described in the following Variety Description Information.It has been self-pollinated a sufficient number of generations withcareful attention to uniformity of plant type. The cultivar has beenincreased with continued observation for uniformity. No variant traitshave been observed or are expected for agronomical important traits inlettuce cultivar SCARAMANGA.

Lettuce cultivar SCARAMANGA has the following morphologic and othercharacteristics (based primarily on data collected in California).Comparison data with similar varieties Blackhawk and Showdown are alsoprovided. The field tests were experimental trials, under supervision ofthe applicant.

TABLE 1 VARIETY DESCRIPTION INFORMATION SCARAMANGA Blackhawk ShowdownPlant: Type Cutting or gathering Cutting or gathering Cutting orgathering red leaf type red leaf type red leaf type Seed: Color WhiteBlack Black Cotyledon to Fourth Leaf Stage: Shape of CotyledonesIntermediate Intermediate Intermediate Shape of fourth leaf ElongatedOval Elongated Length/width index of fourth leaf: 16 12 16 L/W* 10Apical Margin Entire Moderately dentate Finely dentate Basal marginFinely dentate Crenate/Gnawed Crenate/Gnawed Undulation Slight MarkedMarked Antocyanin distribution Margin only Margin only Margin onlyAntocyanin concentration Intense Intense Intense Antocyanin RolingAbsent Present Present Cupping Slight Slight Markedly Reflexing NoneApical margin Lateral Margin Mature Leaves: Margin incision depthAbsent/Shallow Moderate Absent/Shallow Margin indentation EntireShallowly dentate Crenate Undulation of the apical margin ModerateStrong Strong Antocyanin distribution Throughout Throughout naAntocyanin concentration Intense Intense Intense Size Large Medium SmallGlossiness Glossy Moderate Moderate Blistering Moderate Strong ModerateLeaf Thickness Thin Thick Intermediate Trichromes Absent Present presentPlant: Spread of frame leaves 29 cm 31 cm 29 cm Head Diameter 27 cm 28cm 26 cm Head weight 200 g 155 g 70 g Core: Diameter at Base of Head 23mm 13 mm na Ratio of head diameter/core diameter 1.2 2.2 na Core heightfrom base of head to apex 67 mm 43 mm na Bolting (first water date June21st) Number of days from first water date to 70 54 45 seed stalkemergence in summer conditions Height of mature seed stalk 115 cm 112 cm80 cm Spread of bolter plant 46 cm 38 cm 35 cm Bolter leaves StraightCurved Curved Margin Entire Dentate Dentate Bolter habit: terminalinflorescence Present Present Present Bolter habit: lateral shootsPresent Present Present Bolter habit: basal side shoots Absent AbsentAbsent Bremia resistance European Isolates BI1-30 BI1-16, 19, 21, 23BL1-31 California Isolates CA I-VI CA I-VI CA I-VIII

Lettuce cultivar GIMLET has superior characteristics and was developedfrom an initial cross that was made in a greenhouse in Gilroy, Calif.during the first year of development under number 710600116, between twoexperimental Lactuca sativa plants. The F₁ plants were grown in Gilroy,Calif. F2 plants were highly heterogeneous and phenotypic selection onlystarted at the F3 generation for plants showing Bremia resistance. F3were selected for Bremia resistance. The F4 plant was a red leaf plant,similar to the Blackhawk variety but glossier and less blistered.Subsequence generations were selected for the plant, with increasinglevels of uniformity, up to the F8 generation.

Cultivar GIMLET is a red leaf lettuce similar to lettuce cultivarsBlackhawk and Shockwave. While somewhat similar to Blackhawk andShockwave, Cultivar GIMLET has smoother and more glossy leaf that thetwo other varieties. Resistance to Bremia is also improved, Blackhawkbeing resistant to CA I-VI/B11-16, 19, 21, 23 and Shockwave beingresistant to CA I-VI/B11-25 and 27 while GIMLET is resistant to CAI-VIII/B11-26 and 28. Further, Cultivar GIMLET is slower to bolt thaneither of Blackhawk and Shockwave

Cultivar GIMLET is a year-round, mildew resistant, red-leaf lettucevariety suitable for baby-leaf salad production in coastal California,the U.S. desert southwest, northern Europe, and southern Europe. It hasdark red, glossy leaf, and resistance to Bremia lactucae(CAI-VIII:BL1-26 and 28).

Some of the criteria used to select in various generations include:Bremia resistance, slow bolting and red leaf color.

Cultivar GIMLET has shown uniformity and stability for the traits, asdescribed in the following Variety Description Information. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The cultivar has been increasedwith continued observation for uniformity. No variant traits have beenobserved or are expected for agronomical important traits in lettucecultivar GIMLET.

Lettuce cultivar GIMLET the following morphologic and othercharacteristics (based primarily on data collected in California).Comparison date with similar varieties Blackhawk and Shockwave are alsoprovided. The field tests were experimental trials, under supervision ofthe applicant.

TABLE 2 VARIETY DESCRIPTION INFORMATION GIMLET Blackhawk ShockwavePlant: Type Cutting or gathering Cutting or gathering Cutting orgathering red leaf type red leaf type red leaf type Seed: Color WhiteBlack White Cotyledon to Fourth Leaf Stage: Shape of CotyledonesIntermediate Intermediate Intermediate Shape of fourth leaf ElongatedOval Elongated Length/width index of fourth leaf: 18 12 16 L/W* 10Apical Margin Entire Moderately dentate Finely dentate Basal marginEntire Crenate/Gnawed Coarsely dentate Undulation Medium Marked MediumAntocyanin distribution Throughtout Throughtout Throughtout Antocyaninconcentration Intense Intense Intense Antocyanin Roling Absent PresentPresent Cupping Slight Slight Slight Reflexing None Apical marginLateral Margin Mature Leaves: Margin incision depth Absent/ShallowModerate Moderate Margin indentation Entire Shallowly dentate CrenateUndulation of the apical margin Moderate Strong Moderate Antocyanindistribution Throughout Throughout Throughout Antocyanin concentrationIntense Intense Intense Size Small Medium Medium Glossiness GlossyModerate Moderate Blistering Absent Strong Moderate Leaf Thickness ThinThick Intermediate Trichromes Absent Present present Plant: Spread offrame leaves 19 cm 31 cm 33 cm Head Diameter 19 cm 26 cm 28 cm Headweight 65 g 155 g 145 g Core: Diameter at Base of Head 13 mm 13 mm 14 mmRatio of head diameter/core diameter 1.5 2 2 Core height from base ofhead to apex 50 mm 43 mm 45 mm Bolting (first water date June 21st)Number of days from first water date to 70 54 64 seed stalk emergence insummer conditions Height of mature seed stalk 86 cm 112 cm 81 cm Spreadof bolter plant 31 cm 38 cm 40 cm Bolter leaves Curved Curved CurvedMargin Entire Dentate Dentate Bolter habit: terminal inflorescencePresent Present Present Bolter habit: lateral shoots Present PresentPresent Bolter habit: basal side shoots Absent Absent Absent Bremiaresistance European Isolates BI1-26, 28 BI1-16, 19, 21, 23 BI1-25, 27California Isolates CA I-VIII CA I-VI CA I-VI

Lettuce cultivar SPRITZER has superior characteristics and was developedfrom an initial cross that was made in a greenhouse in Gilroy, Calif.during the first year of development under number 71040276, between twoexperimental Lactuca sativa plants, one being a red tango lettuce, theother one being an iceberg lettuce. The F₁ plants were grown in Gilroy,Calif. F2 plants were highly heterogeneous due to the wide originalcross and it took 11 generations of inbreeding to achieve the cultivarSPRITZER, selection criteria at each generation focusing on a tango leafshape, leaf thickness and dark, red color.

Cultivar SPRITZER is a red tango lettuce similar to lettuce cultivarsAsilomar and Intrepid. While somewhat similar to Asilomar and Intrepid,Cultivar SPRITZER has more curled, tango-like leaf leaves than eitherboth of which are classified as red oak-leaf varieties. smoother andmore glossy leaf that the two other varieties. Further, CultivarSPRITZER is faster to bolt than either of Asilomar and Intrepid.Cultivar SPRITZER has also white seed color while Asilomar and Intrepidhave black seed color.

Cultivar SPRITZER is a year-round, mildew resistant, red-tango lettucevariety suitable for baby-leaf salad production in coastal California,the U.S. desert southwest, northern Europe, and southern Europe. It hasresistance to Bremia lactucae (CAI-VIII/BL1-25, 27-28 and 30-31).

Some of the criteria used to select in various generations include:bremia resistance, tango leaf shape, leaf thickness and dark, red color.

Cultivar SPRITZER has shown uniformity and stability for the traits, asdescribed in the following Variety Description Information. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The cultivar has been increasedwith continued observation for uniformity. No variant traits have beenobserved or are expected for agronomical important traits in lettucecultivar SPRITZER.

Lettuce cultivar SPRITZER has the following morphologic and othercharacteristics (based primarily on data collected in California).Comparison date with similar varieties Asilomar and Intrepid are alsoprovided. The field tests were experimental trials, under supervision ofthe applicant.

TABLE 3 VARIETY DESCRIPTION INFORMATION SPRITZER Asilomar IntrepidPlant: Type Red Tango Red oak Red oak Seed: Color White Black BlackCotyledon to Fourth Leaf Stage: Shape of Cotyledones IntermediateIntermediate Intermediate Shape of fourth leaf Pinnately lobed Pinnatelylobed Pinnately lobed Length/width index of fourth leaf: 12 17 16 L/W*10 Apical Margin Entire Crenate/Gnawed Entire Basal margin Lobed LobedLobed Undulation Medium Medium Medium Antocyanin distributionThroughtout Throughtout Throughtout Antocyanin concentration IntenseIntense Intense Antocyanin Roling Absent Absent Absent Cupping UncuppedSlight Uncupped Reflexing None None None Mature Leaves: Margin incisiondepth Deep Deep Deep Margin indentation Shallowly dentate Deeply dentateShallowly dentate Undulation of the apical margin Strong Strong StrongAntocyanin distribution Throughout Throughout Throughout Antocyaninconcentration Intense Intense Intense Size Medium Medium MediumGlossiness Moderate Dull Moderate Blistering Absent Absent Absent LeafThickness Thin Intermediate Thin Trichromes Absent Absent Absent Plant:Spread of frame leaves 18 cm 26 cm 33 cm Head Diameter 16 cm 24 cm 30 cmHead weight 100 g 120 g 180 g Core: Diameter at Base of Head 16 mm 18 mm15 mm Ratio of head diameter/core diameter 1 1.3 2 Core height from baseof head to apex 73 mm 45 mm 65 mm Bolting (first water date June 21st)Number of days from first water date to 56 66 65 seed stalk emergence insummer conditions Height of mature seed stalk 102 cm 89 cm 70 cm Spreadof bolter plant 28 cm 36 cm 21 cm Bolter leaves Straight Curved CurvedMargin Dentate Dentate Dentate Bolter habit: terminal inflorescencePresent Present Present Bolter habit: lateral shoots Present PresentPresent Bolter habit: basal side shoots Absent Absent Absent Bremiaresistance European Isolates BI1-25, 27-28, 30-31 BL1-31 BL1-31California Isolates CA I-VIII CA I-VIII CA I-VIII

Lettuce cultivar MOJITO has superior characteristics and was developedfrom an initial cross that was made in a greenhouse in Gilroy, Calif.during the first year of development under number 71040051, between twoexperimental Lactuca sativa plants, one being a red tango lettuce, theother one being an iceberg lettuce. The F₁ plants were grown in Gilroy,Calif. F2 plants were highly heterogeneous due to the wide originalcross and it took 10 generations of inbreeding to achieve the cultivarMOJITO, selection criteria at each generation focusing on a tango leafshape, leaf thickness and dark, green color.

Cultivar MOJITO is a tango lettuce similar to lettuce cultivars Flurryand Tango. While somewhat similar to Flurry and Tango, Cultivar MOJITOhas darker, narrower, less undulated leaf and higher levels orresistance to Bremia lactucae.

Cultivar MOJITO is a year-round, mildew resistant, red-tango lettucevariety suitable for baby-leaf salad production in coastal California,the U.S. desert southwest, northern Europe, and southern Europe. It hasresistance to Bremia lactucae (CAI-VIII/BL1-26, 28 and 31).

Some of the criteria used to select in various generations include:Bremia resistance, tango leaf shape, leaf thickness and dark, greencolor.

Cultivar MOJITO has shown uniformity and stability for the traits, asdescribed in the following Variety Description Information. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The cultivar has been increasedwith continued observation for uniformity. No variant traits have beenobserved or are expected for agronomical important traits in lettucecultivar MOJITO.

Lettuce cultivar r MOJITO has the following morphologic and othercharacteristics (based primarily on data collected in California).Comparison date with similar varieties Flurry and Tango are alsoprovided. The field tests were experimental trials, under supervision ofthe applicant.

TABLE 4 VARIETY DESCRIPTION INFORMATION MOJITO Flurry Tango Plant: TypeTango Tango Tango Seed: Color Black Black Black Cotyledon to Fourth LeafStage: Shape of Cotyledones Intermediate Intermediate Intermediate Shapeof fourth leaf Elongated Pinnately lobed Pinnately lobed Length/widthindex of fourth leaf: 9 14 15 L/W* 10 Apical Margin Lobed Coarselydentate Lobed Basal margin Moderately dentate Entire Lobed UndulationSlight Marked Marked Green color Medium green Light green Medium greenAntocyanin distribution Absent Absent Absent Antocyanin Roling AbsentAbsent Present Cupping Uncupped Slight Markedly Reflexing None NoneLateral Margins Mature Leaves: Margin incision depth Deep Deep DeepMargin indentation Deeply dentate Deeply dentate Deeply dentateUndulation of the apical margin Strong Strong Strong Green color Mediumgreen Light green Medium green Antocyanin distribution Absent AbsentAbsent Size Medium Medium Medium Glossiness Moderate Moderate ModerateBlistering Absent Absent Moderate Leaf Thickness Intermediate ThickIntermediate Trichromes Absent Absent Present Plant: Spread of frameleaves 23 cm 19 cm 20 cm Head Diameter 21 cm 18 cm 19 cm Head weight 115g 130 g 60 g Butt: Midrib Moderately raised Flattened Moderately raisedCore: Diameter at Base of Head 20 mm 16 mm 19 mm Ratio of headdiameter/core diameter 1.1 1.2 1 Core height from base of head to apex60 mm 41 mm 29 mm Bolting (first water date June 21st) Number of daysfrom first water date to 57 59 33 seed stalk emergence in summerconditions Height of mature seed stalk 102 cm 95 cm 137 cm Spread ofbolter plant 30 cm 34 cm 30 cm Bolter leaves Straight Straight CurvedMargin Dentate Dentate Dentate Color Medium green Medium green Mediumgreen Bolter habit: terminal inflorescence Present Present PresentBolter habit: lateral shoots Present Present Present Bolter habit: basalside shoots Absent Absent Absent Bremia resistance European IsolatesBI1-26, 28, 31 BL1-17, 19, 21-23, None claimed 25, 27, 30-31 CaliforniaIsolates CA I-VIII CA I-VIII None claimed

Further Embodiments of the Invention

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein either the first or second parent lettuce plant is alettuce plant of the line SCARAMANGA, GIMLET, SPRITZER and/or MOJITO.Further, both first and second parent lettuce plants can come fromcultivar SCARAMANGA, GIMLET, SPRITZER and/or MOJITO, respectively. Whenself-pollinated, or crossed with another lettuce cultivar SCARAMANGAplant, the lettuce cultivar SCARAMANGA will be stable, while whencrossed with another, different lettuce cultivar plant, an F₁ hybridseed is produced. When self-pollinated, or crossed with another lettucecultivar GIMLET plant, the lettuce cultivar GIMLET will be stable, whilewhen crossed with another, different lettuce cultivar plant, an F₁hybrid seed is produced. When self pollinated, or crossed with anotherlettuce cultivar SPRITZER plant, the lettuce cultivar SPRITZER will bestable, while when crossed with another, different lettuce cultivarplant, an F₁ hybrid seed is produced When self-pollinated, or crossedwith another lettuce cultivar MOJITO plant, the lettuce cultivar MOJITOwill be stable, while when crossed with another, different lettucecultivar plant, an F₁ hybrid seed is produced. Such methods ofhybridization and self-pollination of the lettuce are well known tothose skilled in the art of lettuce breeding. See, for example, F. A.Bliss, 1980, Common Bean, In Hybridization of Crop Plants, Fehr andHadley, eds., Chapter 17: 273-284, American Society of Agronomy and CropScience Society of America, Publishers.

Still further, this invention also is directed to methods for producingan SCARAMANGA-derived lettuce plant by crossing cultivar SCARAMANGA witha second lettuce plant and growing the progeny seed, and repeating thecrossing and growing steps with the cultivar SCARAMANGA-derived plantfrom 0 to 7 times. Thus, any such methods using the cultivar SCARAMANGAare part of this invention: selfing, backcrosses, hybrid production,crosses to populations, and the like. All plants produced using cultivarSCARAMANGA as a parent are within the scope of this invention, includingplants derived from cultivar SCARAMANGA. Advantageously, the cultivar isused in crosses with other, different, cultivars to produce firstgeneration (F₁) lettuce seeds and plants with superior characteristics.

Still further, this invention also is directed to methods for producingan GIMLET-derived lettuce plant by crossing cultivar GIMLET with asecond lettuce plant and growing the progeny seed, and repeating thecrossing and growing steps with the cultivar GIMLET-derived plant from 0to 7 times. Thus, any such methods using the cultivar GIMLET are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar GIMLET asa parent are within the scope of this invention, including plantsderived from cultivar GIMLET. Advantageously, the cultivar is used incrosses with other, different, cultivars to produce first generation(F₁) lettuce seeds and plants with superior characteristics.

Still further, this invention also is directed to methods for producingan SPRITZER-derived lettuce plant by crossing cultivar SPRITZER with asecond lettuce plant and growing the progeny seed, and repeating thecrossing and growing steps with the cultivar SPRITZER-derived plant from0 to 7 times. Thus, any such methods using the cultivar SPRITZER arepart of this invention: selfing, backcrosses, hybrid production, crossesto populations, and the like. All plants produced using cultivarSPRITZER as a parent are within the scope of this invention, includingplants derived from cultivar SPRITZER. Advantageously, the cultivar isused in crosses with other, different, cultivars to produce firstgeneration (F₁) lettuce seeds and plants with superior characteristics.

Still further, this invention also is directed to methods for producingan MOJITO-derived lettuce plant by crossing cultivar MOJITO with asecond lettuce plant and growing the progeny seed, and repeating thecrossing and growing steps with the cultivar MOJITO-derived plant from 0to 7 times. Thus, any such methods using the cultivar MOJITO are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar MOJITO asa parent are within the scope of this invention, including plantsderived from cultivar MOJITO. Advantageously, the cultivar is used incrosses with other, different, cultivars to produce first generation(F₁) lettuce seeds and plants with superior characteristics.

The invention is also directed to a method of producing a lettuce plantderived from the lettuce SCARAMANGA, the method comprising the steps of:(a) preparing a progeny plant derived from SCARAMANGA, by crossing aplant of SCARAMANGA with a second lettuce plant; (b) crossing theprogeny plant thereof with itself or a second lettuce plant to produce aseed of a progeny plant of a subsequent generation; (c) growing aprogeny plant of a subsequent generation from said seed and crossing theprogeny plant of a subsequent generation with itself or a second lettuceplant; and (d) repeating step b) or c) for at least 1 more generation toproduce a lettuce plant derived from the lettuce SCARAMANGA.

The invention is also directed to a method of producing a lettuce plantderived from the lettuce GIMLET, the method comprising the steps of: (a)preparing a progeny plant derived from GIMLET, by crossing a plant ofGIMLET with a second lettuce plant; (b) crossing the progeny plantthereof with itself or a second lettuce plant to produce a seed of aprogeny plant of a subsequent generation; (c) growing a progeny plant ofa subsequent generation from said seed and crossing the progeny plant ofa subsequent generation with itself or a second lettuce plant; and (d)repeating step b) or c) for at least 1 more generation to produce alettuce plant derived from the lettuce GIMLET.

The invention is also directed to a method of producing a lettuce plantderived from the lettuce SPRITZER, the method comprising the steps of:(a) preparing a progeny plant derived from SPRITZER by crossing a plantof SPRITZER with a second lettuce plant; (b) crossing the progeny plantthereof with itself or a second lettuce plant to produce a seed of aprogeny plant of a subsequent generation; (c) growing a progeny plant ofa subsequent generation from said seed and crossing the progeny plant ofa subsequent generation with itself or a second lettuce plant; and (d)repeating step b) or c) for at least 1 more generation to produce alettuce plant derived from the lettuce SPRITZER.

The invention is also directed to a method of producing a lettuce plantderived from the lettuce MOJITO, the method comprising the steps of: (a)preparing a progeny plant derived from MOJITO, by crossing a plant ofMOJITO with a second lettuce plant; (b) crossing the progeny plantthereof with itself or a second lettuce plant to produce a seed of aprogeny plant of a subsequent generation; (c) growing a progeny plant ofa subsequent generation from said seed and crossing the progeny plant ofa subsequent generation with itself or a second lettuce plant; and (d)repeating step b) or c) for at least 1 more generation to produce alettuce plant derived from the lettuce MOJITO.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, heads, stems, roots, anthers, pistils, root tips, leaves,meristematic cells, axillary buds and the like.

As is well-known in the art, tissue culture of lettuce can be used forthe in vitro regeneration of a lettuce plant. Tissue culture of varioustissues of lettuce and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Teng et al.,HortScience, 27: 9, 1030-1032 (1992), Teng et al., HortScience. 28: 6,669-671 (1993), Zhang et al., Journal of Genetics and Breeding, 46: 3,287-290 (1992), Webb et al., Plant Cell Tissue and Organ Culture, 38: 1,77-79 (1994), Curtis et al., Journal of Experimental Botany, 45: 279,1441-1449 (1994), Nagata et al., Journal for the American Society forHorticultural Science, 125: 6, 669-672 (2000). Thus, it is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of lettuce cultivars ofthe present application.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary budsand the like. Means for preparing and maintaining plant tissue cultureare well known in the art. By way of example, a tissue culturecomprising organs has been used to produce regenerated plants. U.S. Pat.Nos. 5,959,185, 5,973,234, and 5,977,445 describe certain techniques,the disclosures of which are incorporated herein by reference.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed cultivar.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedlettuce plants using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford, et al.,Plant Physiol., 86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86(1987); Svab, et al., Plant Mol. Biol., 14:197 (1990); Hille, et al.,Plant Mol. Biol., 7:171 (1986)). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate or bromoxynil(Comai, et al., Nature, 317:741-744 (1985); Gordon-Kamm, et al., PlantCell, 2:603-618 (1990); and Stalker, et al., Science, 242:419-423(1988)).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells (Chalfie,et al., Science, 263:802 (1994)). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in lettuce. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in lettuce. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See, Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett, et al., Proc. Natl. Acad. Sci. USA,90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Gatz, et al., Mol. Gen. Genetics,243:32-38 (1994)), or Tet repressor from Tn10 (Gatz, et al., Mol. Gen.Genetics, 227:229-237 (1991)). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena,et al., Proc. Natl. Acad. Sci. USA, 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in lettuce or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell 2, 163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genetics, 231:276-285 (1992) and Atanassova, et al., Plant Journal,2 (3):291-300 (1992)). The ALS promoter, Xba1/Nco1 fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/Nco1 fragment), represents a particularly usefulconstitutive promoter. See, PCT Application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in lettuce.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., Proc. Natl. Acad. Sci. USA82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson, et al., EMBO J., 4(11):2723-2729(1985) and Timko, et al., Nature, 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell, et al., Mol.Gen. Genetics, 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 or a microspore-preferred promoter such as that from apg(Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine during protein synthesis and processing where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., etal., “Structure and Organization of Two Divergent Alpha-Amylase Genesfrom Barley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., Proc. Natl. Acad. Sci., 88:834 (1991); Gould,et al., J. Cell. Biol., 108:1657 (1989); Creissen, et al., Plant J.,2:129 (1991); Kalderon, et al., Cell, 39:499-509 (1984); Steifel, etal., “Expression of a maize cell wall hydroxyproline-rich glycoproteingene in early leaf and root vascular differentiation,” Plant Cell,2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a lettuce plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Methods in Plant Molecular Biology and Biotechnology,Glick and Thompson Eds., CRC Press, Inc., Boca Raton, 269:284 (1993).Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example, Jones, et al., Science,266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin, et al., Science, 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); Mindrinos, et al., Cell, 78:1089 (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser, et al., Gene,48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

C. A lectin. See, for example, Van Damme, et al., Plant Molec. Biol.,24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See, PCT Application US93/06487 which teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Molec. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani, etal., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosure of Pratt, et al., Biochem. Biophys. Res. Comm., 163:1243(1989) (an allostatin is identified in Diploptera puntata). See also,U.S. Pat. No. 5,266,317 to Tomalski, et al., which discloses genesencoding insect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule, forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See, PCTApplication WO 93/02197 (Scott, et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also, Kramer, et al., InsectBiochem. Molec. Biol., 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck, et al.,Plant Molec. Biol., 21:673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Molec. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See, PCT Application WO 95/16776, whichdiscloses peptide derivatives of tachyplesin which inhibit fungal plantpathogens, and PCT Application WO 95/18855, which teaches syntheticantimicrobial peptides that confer disease resistance.

M. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci, 89:43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virusand tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilising plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., Bio/Technology,10:1436 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/Technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

S. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., “Plant diseaseresistance. Grand unification system theory in sight,” Current Biology,5(2) (1995).

T. Antifungal genes. See, Cornelissen and Melchers, “Strategies forControl of Fungal Diseases with Transgenic Plants,” Plant Physiol.,101:709-712 (1993); and Bushnell, et al., “Genetic Engineering ofDisease Resistance in Cereal,” Can. J. of Plant Path., 20(2):137-149(1998).

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988), and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotidesequence of a form of EPSPS which can confer glyphosate resistance. ADNA molecule encoding a mutant aroA gene can be obtained under ATCCAccession No. 39256, and the nucleotide sequence of the mutant gene isdisclosed in U.S. Pat. No. 4,769,061 to Comai. European PatentApplication No. 0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374to Goodman, et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. See also, Russel, D. R., et al., Plant Cell Report,12:3 165-169 (1993). The nucleotide sequence of aphosphinothricin-acetyl-transferase (PAT) gene is provided in EuropeanApplication No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/Technology, 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori, et al., “AnAcetohydroxy acid synthase mutant reveals a single site involved inmultiple herbicide resistance,” Mol. Gen. Genet., 246:419-425 (1995).Other genes that confer tolerance to herbicides include a gene encodinga chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochromeP450 oxidoreductase (Shiota, et al., “Herbicide-resistant Tobacco PlantsExpressing the Fused Enzyme between Rat Cytochrome P4501A1 (CYP1A1) andYeast NADPH-Cytochrome P450 Oxidoreductase,” Plant Physiol., 106:17(1994)), genes for glutathione reductase and superoxide dismutase (Aono,et al., “Paraquat tolerance of transgenic Nicotiana tabacum withenhanced activities of glutathione reductase and superoxide dismutase,”Plant Cell Physiol., 36:1687 (1995)), and genes for variousphosphotransferases (Datta, et al., “Herbicide-resistant Indica riceplants from IRRI breeding line IR72 after PEG-mediated transformation ofprotoplants,” Plant Mol. Biol., 20:619 (1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

Increased iron content of the lettuce, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae, 521, 101-109 (2000). Parallel to the improved ironcontent enhanced growth of transgenic lettuce was also observed in earlydevelopment stages.

B. Decreased nitrate content of leaves, for example by transforming alettuce plant with a gene coding for a nitrate reductase. See forexample Curtis et al., Plant Cell Report, 18: 11, 889-896 (1999).

C. Increased sweetness of the lettuce by transferring a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology, 10: 5, 561-564(1992).

D. Delayed senescence or browning by transferring a gene or acting onthe transcription of a gene involved in the plant senescence. See Wanget al. In Plant Mol. Bio, 52:1223-1235 (2003) on the role of thedeoxyhypusine synthase in the senescence. See also U.S. Pat. No.6,538,182 issued Mar. 25, 2003.

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in-vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch, et al., Science,227:1229 (1985); Diant, et al., Molecular Breeding, 3:1, 75-86 (1997).A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteriawhich genetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant Sci., 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by Gruber, et al., supra, Miki, et al., supra, and Moloney, etal., Plant Cell Reports, 8:238 (1989). See also, U.S. Pat. No. 5,591,616issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some cereal or vegetablecrop species and gymnosperms have generally been recalcitrant to thismode of gene transfer, even though some success has been achieved inrice and corn. Hiei, et al., The Plant Journal, 6:271-282 (1994) andU.S. Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation. A generally applicable method of plant transformation ismicroprojectile-mediated transformation where DNA is carried on thesurface of microprojectiles measuring 1 to 4 microns. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al., Pl. Cell. Rep., 12, 165-169 (3 Jan. 1993); Aragao, F. J. L., etal., Plant Mol. Biol., 20, 357-359 (2 Oct. 1992); Aragao, Theor. Appl.Genet., 93:142-150 (1996); Kim, J.; Minamikawa, T., Plant Science,117:131-138 (1996); Sanford, et al., Part. Sci. Technol., 5:27 (1987);Sanford, J. C., Trends Biotech., 6:299 (1988); Klein, et al., Bio/Tech.,6:559-563 (1988); Sanford, J. C., Physiol Plant, 7:206 (1990); Klein, etal., Biotechnology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/Technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985); Christou, et al., Proc Natl. Acad. Sci. USA, 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. Hain, etal., Mol. Gen. Genet., 199:161 (1985) and Draper, et al., Plant CellPhysiol., 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described Saker, M. and Kuhne, T., BiologiaPlantarum, 40(4):507-514 (1997/98); D'Halluin, et al., Plant Cell,4:1495-1505 (1992); and Spencer, et al., Plant Mol. Biol., 24:51-61(1994)).

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic trait thathas been engineered into a particular lettuce line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple X by Y cross or the process ofbackcrossing depending on the context.

Backcrossing

When the term lettuce plant, cultivar or lettuce line are used in thecontext of the present invention, this also includes cultivars where oneor more desired traits has been introduced through backcrossing methods,whether such trait is a naturally occurring one or a transgenic one.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the line. The term “backcrossing” asused herein refers to the repeated crossing of a hybrid progeny back tothe recurrent parent, i.e., backcrossing one, two, three, four, five,six, seven, eight, nine, or more times to the recurrent parent. Theparental lettuce plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental lettuce plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second line (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, generally determined at a 5% significance levelwhen grown in the same environmental condition, in addition to the geneor genes transferred from the nonrecurrent parent. It has to be notedthat some, one, two, three, or more, self-pollination and growing ofpopulation might be included between two successive backcrosses. Indeed,an appropriate selection in the population produced by theself-pollination, i.e., selection for the desired trait andphysiological and morphological characteristics of the recurrent parentmight be equivalent to one, two or even three, additional backcrosses ina continuous series without rigorous selection, saving time, money andeffort to the breeder. A non limiting example of such a protocol wouldbe the following: (a) the first generation F₁ produced by the cross ofthe recurrent parent A by the donor parent B is backcrossed to parent A;(b) selection is practiced for the plants having the desired trait ofparent B; (c) selected plants are self-pollinated to produce apopulation of plants where selection is practiced for the plants havingthe desired trait of parent B and the physiological and morphologicalcharacteristics of parent A; (d) the selected plants are backcrossedone, two, three, four, five, six, seven, eight, nine, or more times toparent A to produce selected backcross progeny plants comprising thedesired trait of parent B and the physiological and morphologicalcharacteristics of parent A. Step (c) may or may not be repeated andincluded between the backcrosses of step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a gene or genes of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicalimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a single geneand dominant allele, multiple genes and recessive allele(s) may also betransferred and therefore, backcross breeding is by no means restrictedto character(s) governed by one or a few genes. In fact the number ofgenes might be less important than the identification of thecharacter(s) in the segregating population. In this instance it may thenbe necessary to introduce a test of the progeny to determine if thedesired characteristic(s) has been successfully transferred. Such testsencompass visual inspection, simple crossing but also follow up of thecharacteristic(s) through genetically associated markers and molecularassisted breeding tools. For example, selection of progeny containingthe transferred trait is done by direct selection, visual inspection fora trait associated with a dominant allele, while the selection ofprogeny for a trait that is transferred via a recessive allele requiresselfing the progeny to determine which plant carries the recessiveallele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic. An example of a gene controlling resistance to the lettuceleaf aphid Nasonovia ribisnigri (Nr gene) can be found in Van der Arendand Schijndel in Breeding for Resistance to insects and Mites, IOBC wprsBulletin 22(10), 35-43 (1999). These genes are generally inheritedthrough the nucleus. Some other single gene traits are described in U.S.Pat. Nos. 5,777,196, 5,948,957, and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

In 1981 the backcross method of breeding accounted for 17% of the totalbreeding effort for inbred corn line development in the United States,according to, Hallauer, A. R., et al., “Corn Breeding,” Corn and CornImprovement, No. 18, pp. 463-481 (1988).

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,Principles of Plant Breeding, published by John Wiley & Sons, Inc.) Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a variety with exactly the adaptation, yielding ability, andquality characteristics of the recurrent parent but superior to thatparent in the particular characteristic(s) for which the improvementprogram was undertaken. Therefore, this method provides the plantbreeder with a high degree of genetic control of his work.

The backcross method is scientifically exact because the morphologicaland agricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method,” Jour. Amer. Soc. Agron.,22:289-244 (1930)).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart’ wheat and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in ‘CaliforniaCommon’ alfalfa to create ‘Caliverde’. This new ‘Caliverde’ varietyproduced through the backcross process is indistinguishable from‘California Common’ except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics, and simply inherited quantitative characters,such as earliness, plant height, and seed size and shape. In thisregard, a medium grain type variety, ‘Calady’, has been produced byJones and Davis. As dealing with quantitative characteristics, theyselected the donor parent with the view of sacrificing some of theintensity of the character for which it was chosen, i.e., grain size.‘Lady Wright’, a long grain variety was used as the donor parent and‘Coloro’, a short grain variety as the recurrent parent. After fourbackcrosses, the medium grain type variety ‘Calady’ was produced.

Deposit Information

A deposit of the lettuce seeds of this invention is maintained byShamrock Seed Company Inc., 3 Harris Place, Salinas, Calif. 93901-4593USA. In addition, a sample of the lettuce seeds of this invention hasbeen deposited with the National Collections of Industrial, Food andMarine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB243RY, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited lettuce cultivar SPRITZER(deposited as NCIMB Accession No. 42641):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. Upon granting of the patent the strain will be available to thepublic under conditions specified in 37 CFR 1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the enforceable lifeof the patent, whichever is longer;

4. The viability of the biological material at the time of deposit willbe tested; and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A seed of lettuce cultivar designated SPRITZER,wherein a representative sample of seed of said cultivar has beendeposited under NCIMB No.
 42641. 2. A lettuce plant, or a part thereof,produced by growing the seed of claim
 1. 3. A tissue culture ofregenerable cells produced from the plant of claim 2 wherein said cellsof the tissue culture are produced from a plant part selected from thegroup consisting of embryos, meristematic cells, leaves, pollen, root,root tips, stems, anther, pistils, axillary buds, flowers and seeds. 4.A lettuce plant regenerated from the tissue culture of claim 3, saidplant having the morphological and physiological characteristics oflettuce cultivar SPRITZER, wherein a representative sample of seed hasbeen deposited under NCIMB No.
 42641. 5. A method for producing alettuce seed comprising crossing a first parent lettuce plant with asecond parent lettuce plant and harvesting the resultant hybrid lettuceseed, wherein said first parent lettuce plant or second parent lettuceplant is the lettuce plant of claim
 2. 6. A hybrid lettuce seed producedby the method of claim
 5. 7. A method for producing an herbicideresistant plant comprising transforming the lettuce plant of claim 2with a transgene that confers herbicide resistance to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine, and benzonitrile.8. An herbicide resistant lettuce plant, or a part thereof, produced bythe method of claim
 7. 9. A method for producing an insect resistantlettuce plant comprising transforming the lettuce plant of claim 2 witha transgene that confers insect resistance.
 10. An insect resistantlettuce plant, or a part thereof, produced by the method of claim
 9. 11.A method for producing a disease resistant lettuce plant comprisingtransforming the lettuce plant of claim 2 with a transgene that confersdisease resistance.
 12. A disease resistant lettuce plant, or a partthereof, produced by the method of claim
 11. 13. A method of producing alettuce plant derived from the lettuce SPRITZER, the method comprisingthe steps of: (a) preparing a progeny plant derived from SPRITZER bycrossing the plant of claim 2 with a second lettuce plant; (b) crossingthe progeny plant with itself or a second lettuce plant to produce aseed of a progeny plant of a subsequent generation; (c) growing aprogeny plant of a subsequent generation from said seed and crossing theprogeny plant of a subsequent generation with itself or a second lettuceplant; and (d) repeating step b) or c) for at least 1 more generation toproduce a lettuce plant derived from the lettuce SPRITZER.
 14. A lettucehead, produced by growing the seed of claim
 1. 15. A lettuce plant, or apart thereof, having all the physiological and morphologicalcharacteristics of lettuce cultivar SPRITZER listed in Table
 3. 16. Alettuce plant, or a part thereof, having the physiological andmorphological characteristics of lettuce cultivar SPRITZER, wherein arepresentative sample of seed of said cultivar has been deposited underNCIMB No.
 42641. 17. A method for producing lettuce cultivar SPRITZERseed comprising crossing a first parent lettuce plant with a secondparent lettuce plant and harvesting the resultant lettuce seed, whereinboth said first and second lettuce plants are the lettuce plant of claim16.
 18. A method of introducing a desired trait into lettuce cultivarSPRITZER comprising: (a) crossing a lettuce cultivar SPRITZER plantgrown from lettuce cultivar SPRITZER seed, wherein a representativesample of seed has been deposited under NCIMB No. 42641, with anotherlettuce plant that comprises a desired trait to produce F₁ progenyplants, wherein the desired trait is selected from the group consistingof insect resistance, disease resistance, water stress tolerance, heattolerance, improved shelf life, delayed senescence, and improvednutritional quality; (b) selecting one or more progeny plants that havethe desired trait to produce selected progeny plants; (c) crossing theselected progeny plants with the lettuce cultivar SPRITZER plants toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait and physiological and morphologicalcharacteristics of lettuce cultivar SPRITZER listed in Table 3 toproduce selected backcross progeny plants; and (e) repeating steps (c)and (d) three or more times in succession to produce selected fourth orhigher backcross progeny plants that comprise the desired trait and thephysiological and morphological characteristics of lettuce cultivarSPRITZER listed in Table
 3. 19. A lettuce plant produced by the methodof claim 18, wherein the plant has the desired trait and thephysiological and morphological characteristics of lettuce cultivarSPRITZER listed in Table 3 when grown under the same environmentalconditions.
 20. The lettuce plant of claim 19, wherein the desired traitis herbicide resistance and the resistance is conferred to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine, and benzonitrile.21. The lettuce plant of claim 19, wherein the desired trait is insectresistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.
 22. The lettuce plant ofclaim 19, wherein the desired trait is selected from the groupconsisting of insect resistance, disease resistance, water stresstolerance, heat tolerance, improved shelf life, delayed senesence, andimproved nutritional quality.